1. Field of the Invention
The present invention relates to novel plasmids useful as cloning and production vectors in the field of recombinant DNA technology, the replication behaviour of which is uncontrolled under conditions favourable therefor, and to a method for preparing such plasmids.
2. Description of the Related Art
Plasmids with conditional uncontrolled replication behaviour (so-called runaway plasmids) are known from, e.g., published European Patent Application No. 78101877.5. The replication of these plasmids is temperature-dependent in that the plasmids show a controlled, constant, low copy number when host bacteria carrying the plasmids are grown at one temperature, e.g. a temperature about 30.degree. C., and an uncontrolled copy number when the host bacteria are grown at a different temperature, e.g. a temperature above about 36.degree. C.
The plasmids disclosed in the above-mentioned patent application were isolated by chemical mutagenisation of plasmid R1, a wild-type plasmid which is known to replicate autonomously in Escherichia coli. The mutagenisation was performed in two stages, the first stage comprising the isolation of a plasmid-carrying bacterial clone in which the plasmid copy number was about 1-2 at 30.degree. C. and 4-5 times higher at 40.degree. C. and the second stage comprising the isolation of a bacterial clone in which the plasmid showed an uncontrolled copy number at the higher temperature. The plasmids showing runaway behaviour were thus double mutants of the parent plasmid.
The above-mentioned patent application also discloses miniplasmids which are useful as cloning vectors because they are relatively easily transformed to host bacteria due to their small size. These miniplasmids have retained the genes responsible for the temperature-dependent runaway replication behaviour.
As cloning and production vectors the plasmids disclosed in the above-mentioned patent application may be used to obtain gene products of inserted fragments of foreign DNA. The temperature-dependent replication of the plasmids may be utilized to produce amplified amounts of gene products from DNA fragments of, for instance, eucaryotic origin. Such amplification of gene product has not been available with conventional plasmid DNA amplification techniques which work by, for instance, the addition of chloramphenicol to the culture medium, as the presence of chloramphenicol causes the protein synthesis to stop.
The plasmids disclosed in the above-mentioned patent application may advantageously be used as cloning and production vectors in cases where an inserted foreign gene codes for a product which is detrimental to the bacterium to which the plasmids are transformed as, at low temperatures, the plasmids have a low copy number and, consequently, little gene expression. This means that during the multiplication stage of the culture which is conducted at low temperatures the bacteria are not likely to be impaired.